Pharmacology & Pharmacy, 2017, 8, 90-108 http://www.scirp.org/journal/pp

ISSN Online: 2157-9431 ISSN Print: 2157-9423

DOI: 10.4236/pp.2017.83007 March 31, 2017

The Beneficial Effects of Oral Cotrimoxazole upon Likely Biomarkers of Oxidative Stress in Advanced Fibrotic Lung Disease

Veronica Varney1*, David Salisbury1, Helen Parnell1, Siva Ratnatheepan1, Alex Nicholas2, Ginny Quirke1, Nazira Sumar2, Amolak Bansal2

1Department of Respiratory Medicine, St Helier Hospital, London, UK 2Department of Immunology, St Helier Hospital, London, UK

Abstract Introduction: In cases of idiopathic pulmonary fibrosis, we have observed an elevation in mean red cell volume, serum gamma glutamyl transferase and pe-ripheral monocyte counts, initially in a pilot study but also in new incident cases. These changes could not be explained by drug therapy, vitamin defi-ciency or other diseases. Method: We compared the peripheral blood abnor-malities in 149 patients with lung fibrosis to 448 age and sex matched con-trols. We also examined the effect of cotrimoxazole treatment for 12 weeks on these abnormalities. From the pilot study of cotrimoxazole in lung fibrosis pa-tients, the relationship of the peripheral blood monocyte count and serum cytokine transforming growth factor beta-1 was examined. Epstein Barr viral status was examined in a selection of patients in case it explained our observa-tions. Results: The findings confirm the elevation in mean red cell volume, gamma glutamyl transferase and peripheral monocyte counts in patients compared with matched controls. Oral cotrimoxazole ameliorated these 3 blood abnormalities. Serological evidence of Epstein Barr viral infection was present in tested patients but active viral replication was absent. The mono-cyte count had a linear relationship with the serum transforming growth fac-tor beta-1 levels, which increased by 600 pg/ml for every of 0.1 × 109/l increase in the monocyte count. Conclusion: These observations may reflect oxidative stress which was reduced by cotrimoxazole. A related sulphonamide “dap-sone” is known to reduce oxidative stress through direct effects on neutrophil and mo-nocyte function; similar effects may explain these findings and re-quire a formal study.

How to cite this paper: Varney, V., Salisbury, D., Parnell, H., Ratnatheepan, S., Nicholas, A., Quirke, G., Sumar, N. and Bansal, A. (2017) The Beneficial Effects of Oral Cotrimoxazole upon Likely Biomar- kers of Oxidative Stress in Advanced Fibro- tic Lung Disease. Pharmacology & Pharma- cy, 8, 90-108. https://doi.org/10.4236/pp.2017.83007 Received: February 3, 2017 Accepted: March 28, 2017 Published: March 31, 2017 Copyright © 2017 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/

Open Access

V. Varney et al.

91

Keywords Oxidative Stress, Cotrimoxazole, Idiopathic Pulmonary Fibrosis, Monocytes, Gamma Glutamyl Transferase, Transforming Growth Factor Beta 1

1. Introduction

In 2008, we published our pilot study examining the effects of oral cotrimoxa-zole upon lung function, exercise capacity and quality of life scores in patients with advanced fibrotic lung disease who were treated with oral cotrimoxazole 960 mg BD and folic acid 5 mg 3 times a week [1].

During the selection of these patients, their blood tests’ pre-randomisation showed three unexpected peripheral blood abnormalities with most patients dis-playing all three. The abnormalities were:

1) Raised red cell mean cell volume (MCV) on the peripheral blood count. 2) Raised serum gamma glutamyl transferase levels (GGT). 3) A raised peripheral blood monocyte count (Mo). These abnormalities could not be explained by past or present medication or

vitamin (B12/folate) deficiency. The elevation in GGT was isolated without ex-planation from alcohol intake, gallstones and past hepatitis (viral, auto-immune or drugs). All abdominal ultrasounds were returned as normal. The monocytosis was persistent, without any viral illness or active Epstein Barr Virus; nor other explanation and the blood film appeared normal.

Scrutiny of all newly presenting patients with idiopathic pulmonary fibrosis (IPF), revealed the same patterns at the initial diagnosis.

Literature searches have revealed data on lung oxidative stress and the me-tabolism of glutathione which could suggest a potential link for all three abnor-malities as biomarkers of lung oxidative stress [2] [3] [4].

Glutathione (GSH) is the most abundant antioxidant in human lung and an important protector of the biomembrane [5] [6]. Levels in lung fluid are 140 times the plasma level where it functions to protect the lung against oxidative stress [4]. Clinical observations show that lung epithelial lining fluids from sub-jects with usual interstitial pneumonitis/idiopathic pulmonary fibrosis (UIP/IPF) were depleted of reduced glutathione, suggesting a redox imbalance (oxidant/ anti-oxidant balance) in this disease [3]. This was shown to be improved by oral N-acetyl cysteine (NAC) which can generate glutathione, and led to the NAC clinical trials [7] [8]. Early published studies of this drug showed the vital capac-ity and transfer factors for carbon monoxide to diminish less in the NAC group compared with placebo at 12 months.

GGT is a plasma membrane ecto-enzyme that plays a key role in glutathione metabolism [9] [10] [11]. GGT catalyses the breakdown of glutathione to gluta-mate and cysteinyl glycine. These are then transported back into the cell for re-synthesis of glutathione. This is critical to prevent oxidative injury [12] [13]. While GGT has been considered an anti-oxidant, recent studies suggest that

V. Varney et al.

92

GGT is up-regulated in response to oxidative stress and GGT cleavage of glu-tathione induces Reactive Oxygen Series (ROS) production [14] [15] [16]. In mice, the genetic absence of GGT (GGT-/-) protects the lung against bleomycin induced lung fibrosis through maintained glutathione levels [17].

In our present study, we compare our peripheral blood findings in patients with UIP/IPF or fibrotic nonspecific interstitial pneumonitis (NSIP) or mixed patterns with age and sex matched controls. We also examined the effect of cotrimoxazole treatment upon these parameters and the relationship between the peripheral blood monocyte count and the serum TGF beta-1 levels in the pilot study patients. A sample of patients had their Epstein Barr viral (EBV) status checked for active viral copy numbers in case EBV reactivation was related to the blood changes.

2. Methods

Study objectives: 1) To assess the 3 peripheral blood abnormalities in 149 new cases of IPF (UIP

or fibrotic NSIP or mixed UIP/NSIP) and compare with 448 age and sex matched healthy controls measured in the same laboratory.

2) To examine the effects of cotrimoxazole treatment on the 3 parameters for all 73 treated patients.

3) To examine changes in the 3 blood parameters (red cell MCV, GGT and monocyte count) on time course graphs in response to cotrimoxazole treatment over 12 weeks from the pilot study data (2 weekly blood tests).

4) To examine the relationship between the peripheral blood monocyte count and the serum transforming growth factor beta 1 (TGF beta-1) levels in the pilot study data.

5) To examine EBV status including active viral copy numbers in a selection of patients.

The pilot study of cotrimoxazole was approved by the London-Surrey borders regional ethics committee 1999 [1] with expansion and reapproved by the same committee for a further study 2006, ISRCT80334919.

2.1. Blood Analysis

Each patient or control had their mean red cell volume and monocyte count meas-ured by the Advia 120, BAYER Haematology systems Auto analyser. The GGT and liver function tests were measured in the Biochemistry department using an Advia 2400 Bayer chemistry systems. The GGT measurement uses synthetic gamma-glu- tamyl p nitroanilide and glycyl glycine as an accelerator for the gamma-glutamyl residue, from which p-nitroaniline is liberated. This liberated product has absorp-tion near 400 nm and the rate of formation is measured photometrically at 410 nm. The advia 2400 method for serum GGT confirms also that it measures GGT in-volved in aspects of glutathione metabolism [18] in the biochemical data.

2.2. Serum Transforming Growth Factor Beta-1 (TGF Beta-1)

Serum TGF beta-1 was measured by Elisa kit supplied by R and D systems, Ab-

V. Varney et al.

93

ingdon, Oxford, UK. This kit had specificity for TGF beta-1, TGF beta-1.2 in the serum with a detection level of 0.25 ng/ml. Samples were measured in duplicate. Coefficient of variation for assay was 4% - 7% for repeat measurements.

2.3. EBV Serology

The Trinity Biotech CaptiaTM Epstein-Barr Viral Capsid Antigen (EBV VCA (P-18)) IgG kit is an Enzyme-Linked Immunosorbent Assays (ELISA) for the qualitative determination of IgG antibodies in human serum to capsid and nu-clear antigens which can indicate past infections with other Epstein-Barr as an aid in the diagnosis of infectious mononucleosis.

2.4. Epstein Barr Active Viral Copy Number Assessment (Table 1 and Table 2)

Quantitation/detection of EBV DNA was carried out at Micropathology Ltd. by nested real-time PCR as follows: DNA was extracted from 200 microliters spe- cimen using a Qiagen MDx Bio-Robot running the QIA amp DNA Blood kit/protocol and was eluted in 200 microliters extraction buffer. 20 microliters of DNA extract from each specimen was subjected to 25 cycles of PCR using an outer primer set targeting the EBV nuclear antigen 1 (EBNA-1) gene. 1 microli-ter of amplicon from this PCR was used as template for a further 25 cycles of real-time PCR using an inner primer set, which was carried out using a Roche Light Cycler 480. PCR products were analysed by melt curve analysis. Where EBV DNA was detected this was quantified with respect to appropriate standards.

2.5. The Fibrosis Patients (Table 1 and Table 2)

A total of 149 new patients presenting clinical features of IPF (mean age 72, range 41 - 92 yrs, and 52% male) were prospectively identified from our respira-tory clinics. Their diagnosis followed the guidelines at their time of selection (ATS/ERS consensus statement and British Thoracic Society 2008) [19] [20]. Pa-tients under 50 yrs usually had a lung biopsy confirmation (5 cases).

Baseline blood results were documented (including B12 and folate, liver func-tion, GGT and FBC) prior to any treatment along with review of their medical notes, clinical examination, medication, lung function, X rays and CT scans. No patients were taking NAC at the time of this data collection. Their HRCT scans were classified according to the method of Hansel and Wells by multidiscipli-nary (MDT) radiological review [21]. Current smokers were excluded from the analysis along with patients with significant emphysema on their HRCT scan.

2.6. The Controls (Table 1)

448 age and sex matched controls were selected prospectively during the same time period. The control group were selected from a large group of patients attending for their pre-operative assessment for day surgery. Patients under the day surgery unit must be judged healthy without any significant cardio-respiratory diseases

V. Varney et al.

94

Table 1. Demographics of cases and controls and inclusion criteria.

Demographics IPF cases Pre-operative day-case controls

No of cases 149 448

% male 52 58

Mean age and range 72 (41 - 92) 73 (40 - 80)

Caucasian 81% 75%

Smoking status 90% ex-smoker Non-smoker or <10 pack years

Chest X ray Abnormal normal

Normal ECG Variable 100%

Past cardiorespiratory disease Yes None

Diabetes 33% None

Alcohol intake units/week 0 - 6 0 - 1

Age match 35 - 40 yrs 45 - 54 yrs 55 - 64 yrs 65 - 75 yrs

75 yrs+

Number of cases-149 2

10 20 40 77

Number of cases-448 40 70 106 110 122

Criteria for IPF cases (major and minor criteria)

as per ATS/ERS criteria

Insidious onset Bibasilar inspiratory crackles

Progressive dyspnoea Duration > 3 months

CT consistent with UIP/NSIP or mixed ± lung biopsy

Restrictive pulmonary function of reduced VC, FVC, TLCO, KCO

Exclusion of other causes of interstitial lung disease

that could preclude day-case anaesthesia and early discharge post-operatively. These assessments included blood tests, ECG and chest x rays, history and ex-amination and medication check by the supervising specialist nursing sister. Non-smoking patients or ex-smokers (<10 pack years) without significant alco-hol intake and a normal chest X ray were selected if of suitable age and sex for our control group. Their pre-operative blood tests were measured in the same laboratory which offered the best possible match for the IPF patients.

2.7. The Effects of Cotrimoxazole Treatment on the 3 Blood Parameters at 12 Weeks (Table 3)

All 73 patients who received cotrimoxazole (46 male, 27 female) had their pre- treatment blood tests compared after 12 weeks (cotrimoxazole 960 mg BD and folic acid 5 mg 3 times a week).

The pilot study: cotrimoxazole time course studies the change in MCV, GGT and Mo has been plotted from the pilot study data (where it was collected double-

V. Varney et al.

95

Table 2. Peripheral blood markers (means ±SD) for MCV (mean cell volume), GGT (gamma glutamyl transferase) and Mo (monocyte count) in controls and fibrosis patients.

Parameters (normal range)

MCV (84-98 fL)

P Value Vs C

GGT (7-40U/L)

P Value Vs C

Mo (<0.6 x109/L)

P Value Vs C

Controls (C) n = 448

mean ± SD 88 ± 4.4 23±13 0.44 ± 0.2

Fibrosis (all cases) n = 149

mean ± SD 93.4 ± 6.7 <0.0001 53.0 ± 38 <0.009 0.64 ± 0.2 <0.0001

UIP n = 104

mean ± SD 93 ± 6.8 <0.0001 54.6 ± 29 <0.0001 0.65 ± 0.2 <0.0001

NSIP n = 31

mean ± SD 93 ± 7.7 <0.0001 38.2 ± 26.6 <0.0001 0.60 ± 0.2 <0.0001

Mixed UIP/NSIP n = 14

mean ± SD 94.4 ± 5.3 <0.0001 44.8 ± 29 <0.0001 0.68 ± 0.2 <0.0001

blind) every 2 weeks in the active n = 10 and placebo n = 10 groups.

Monocyte/TGF beta-1 regression analysis TGF beta-1 levels (measured only in the pilot study) and the peripheral mo-

nocyte counts were examined by linear regression. The peripheral monocyte count (pre-randomisation n = 20) were compared with the TFG beta-1 serum levels from the same blood sample to examine the relationship between mono-cyte count and TGF beta-1 levels.

2.8. Statistics

2.8.1. Comparison of Controls and Fibrosis Patients The fibrosis patients (n = 149) had their 3 blood parameters compared with the age and sex matched controls blood results (n = 448) by unpaired t tests. All the variables were tested for normality using the Shapiro-Wilk W test for normal data. Given the multiple testing procedures, the Bonferroni correction has been applied making the reference level for statistical significance 0.016 for these com-parisons. This included subgroup analysis for UIP and fibrotic NSIP patients.

2.8.2. Assessment of Cotrimoxazole Treatment at 12 Weeks Blood results for the 73 patients treated with cotrimoxazole were compared at 12 weeks using a paired t test. All the variables demonstrated normality using the Shapiro-Wilk W test for normal data, permitting a paired t-test for the differ-ences in pre and 12 week means if normality applied. Identical p values were ob-tained by Wilcoxon-Mann-Whitney test for the difference in the distributions of the pre-and 12 week measurements. Given the multiple testing procedures, the Bonferroni correction was applied and the reference level for statistical signifi-cance has been dropped to 0.05/3 = 0.016.

V. Varney et al.

96

Table 3. The effects of cotrimoxazole in lung fibrosis on the peripheral blood changes showing means ± SD before treatment and at 12 weeks in ILD MCV (mean cell volume), GGT (gamma glutamyl transferase), Mo (monocyte).

Parameters (normal range)

MCV 84 - 98 fL

p Value Pre-post

treatment

GGT 7 – 40 U/L

p Value Pre-post

treatment

Mo <0.6 × 109/L

p Value Pre-post

treatment

pre-treatment n = 73 (UIP/NSIP)

mean ± SD 93.7 ± 6.5 49.6 ± 35 0.67 ± 0.2

cotrimoxazole treated week 12

n = 73 (UIP/NSIP) mean ± SD

90.1 ± 5.0 <0.0001 30.6 ± 31 <0.0011 0.44 ± 0.2 <0.0001

UIP patients pre-treatment

n = 53 mean ± SD

94.3 ± 6.1 51.04 ± 37 0.65 ± 0.2

UIP patients Week 12

cotrimoxazole n = 53

mean ± SD

90.5 ± 4.8

<0.0001 31.1 ± 31 <0.0076 0.430.2 <0.0001

NSIP patients pre-treatment

n = 20 mean ± SD

92.5 ± 7.4 46.1 ± 21 0.72 ± 0.3

NSIP patients week 12 week

co-trimoxazole n = 20

mean ± SD

88.1 ± 5.4 <0.0001 29.4 ± 31 <0.0001 0.45 ± 0.2 <0.0001

Pilot study graphs the 2 weekly group means for the10 active and 10 placebos

were calculated and compared for each time point by Mann Whitney U test with significance at the 5% level. With comparison of baseline measurements (week 0 with week 12) were made by unpaired t test. Graphs were plotted by Graph Pad prism for MCV, GGT and Mo.

2.8.3. Assessment of Monocytes and TGF Beta-1 by Linear Regression TGF beta-1 values in pg/ml and monocyte counts (109/L) were plotted by Graph Pad Prism computer software. The line of best fit and 95% prediction intervals showed by the dotted lines are shown along with calculation of the slope. The p value is displayed on the graph with significance at the 5% level. The correlation coefficient was calculated from the regression line.

3. Results Section

The findings are displayed in tables and figures as described below.

3.1. Peripheral Blood Parameters

Table 2 shows the laboratory units, normal ranges, means ± SD with p values for

V. Varney et al.

97

the 3 blood measurements in the age/sex matched controls (n = 448) and fibrosis patients (n = 149). There were 3 matched controls for each fibrosis patient over the age of 65 yrs and 4 matched controls for those less than 65 yrs of age. The age ranges and percentage of males were well matched.

Mean volume cell was lower in the healthy control group 88fL (95% CI 87.9 - 88.9) compared with the fibrosis group 93.41fL (95% CI 92.8 - 95.4) p < 0.0001, including fibrotic subtypes (Table 2).

Gamma Glutamyl transferase (Table 2) The mean GGT level for the healthy control group was 23U/L (95% CI 21.7 -

24.3), compared with the fibrosis group mean 53 U/L (95% CI 41.9 - 64.7) p < 0.009. Peripheral monocytes (Table 2) Mean Mo counts in the healthy controls were

0.44 × 109/L (95% CI 0.42 - 0.46), with fibrosis group mean 0.64 × 109/L range (95% CI 0.59 - 0.68), p < 0.0001.

3.2. Table 3 Analysis of All Fibrosis Patients Treated with Cotrimoxazole Treatment (n = 73)

Mean MCV (Table 3) pre-treatment was 93.7 fL (95% CI 92.2 - 95.2), and fell to 90.1 fL (95% CI 88.9 - 91.3) after 12 weeks of treatment p < 0.0001. Findings were similar for the subgroups. Statistically significant differences between pre and post means of 3.30 (95% CI 2.43 - 4.77) were obtained.

GGT levels (Table 3) pre-treatment means were 49.6 U/L (95% CI 35.1 - 64.2) and fell by 40% to 30 U/L (95% CI 23.3 - 38.0) after 12 weeks of cotrimoxazole treatment, p < 0.0076.

The distributions of both pre-treatment GGT and post-treatment GGT values exhibit strong departures from normality (p < 0.0001) and remains so even after log transformation. Therefore, the Wilcoxon-Mann-Whitney test reveals strong evidence against the null hypothesis (p < 0.0001).

Monocytes (Mo) Table 3 Peripheral monocytes showed a pre-treatment mean count of 0.67 × 109 /L

(95% CI 0.61 - 0.74) which fell by 35% to 0.44 × 109/L (95% CI 0.38 - 0.50) at 12 weeks p < 0.0001. This was similar for the subtypes analysis (UIP reduced by 34%, fibrotic NSIP 37%) p < 0.0001 respectively.

The distributions of both pre-treatment Mo and post-treatment Mo counts exhibit strong departures from normality (p < 0.0001) but after log transforma-tion they were consistent with normal distributions (p > 0.05). The Wilcoxon- Mann-Whitney test applied to the original scale data (p < 0.0001) demonstrated a difference in the pre and post Mo distributions. This is confirmed by the paired t-test applied to the log transformed data (p < 0.0001) which quantify the difference between the means of the log transformed variables: mean difference (pre-post) = 0.50 (95% CI 0.37 - 0.62).

3.3. Pilot Study Time Course Data for Peripheral Blood Markers

Pilot study Time Course graphs: MCV (Figure 1) shows the group means ± SEM for week 0 - 12 for the 10 active and 10 placebo treated patients. The pla-

V. Varney et al.

98

cebo group shows no consistent change, while the active group shows a decline which is significant after week 6. Comparison of week 0 with week 12 for the ac-tive group confirms a significant reduction p < 0.0001 which is not seen with the placebo group p = 1.0.

Time course Graphs: GGT (Figure 2) shows the group means ± SEM. The placebo group shows no consistent change p = 0.97. The active group decreases significantly over the 12 weeks (p < 0.0145).

Time course graphs: Mo (Figure 3) shows the group means ± SEM. The pla-cebo group shows no change (week 0 - 12) p = 0.64, while the active group de-clines from a mean of 0.9 × 109/l to 0.35 × 109/l (p < 0.003). At week 0 the Mo count is significantly above the placebo p = 0.036, while at 12 weeks it is signifi-cantly below p = 0.035.

3.4. Monocytes and the Relationship to Serum Transforming Growth Factor Beta-1 from Pilot Study Data: Figure 4

Linear regression analysis with the line of best fit and 95% prediction intervals are shown for all pre-randomised patients in the pilot study (n = 20). This gave a slope of 5101 ± 766 p < 0.0001 with a correlation coefficient of r = +2.1. TGF beta-1 levels increased by 600 pg/ml for every 0.1 × 109/L increase in peripheral blood Mo count.

After 12 weeks the active group (n = 10) Mo counts were reduced (Figure 3), but the relationship and slope of monocytes to TGF beta-1 levels remained un-changed and significant (p = 0.0165, slope 4851 ± 1606).

For the placebo treated group at 12 weeks, the relationship between monocyte count and TGF beta-1 remained identical to pre-randomisation and significant (slope 5190 ± 1226 p < 0.002). Data are not shown.

Figure 1. Group means ± SEM for mean cell volume (MCV) measured 2 weekly in the 10 active and 10 placebo treated patients from the original 12 weeks pilot study.

V. Varney et al.

99

Figure 2. Group means ± SEM for gamma glutamyl transferase (GGT) measured 2 weekly in the pilot study (10 active and 10 placebos) over 12 weeks. SEM is show on the graph and the 95% confidence intervals in the table below.

Figure 3. Group means ± SEM for peripheral monocyte counts (Mo) measured 2 weekly in the pilot study (10 active, 10 placebos) over 12 weeks of cotrimoxazole treatment.

TGF beta-1 levels (active group) were 4394 pg/ml and fell to 3633 ng/ml at

week 12 (p = ns). In the placebo group 3387 pg/ml at start and 3115 ng/ml at 12 weeks (p = ns) with no difference found between the active and placebo groups at 12 weeks.

EBV virology screens; All patients with IPF were positive EBV capsid antigen

V. Varney et al.

100

and nuclear IgG. 30 patients were examined for active viral DNA copy numbers in their white cells but none showed evidence active replication.

Figure 5, a schematic diagram of the likely mechanisms of oxidative stress and its possible relationship to glutathione metabolism and the 3 observed blood changes.

4. Discussion The underlying trigger for IPF and fibrotic NSIP is not yet recognised, but oxidative

Figure 4. Linear regression analysis for the relationship between pre-treatment Monocyte counts and the corresponding serum TGF-beta1 measurements for the pilot patients (n = 20). The dotted lines show the 95% prediction intervals of the regression line. The for-mula for the calculation of the linear regression by prism is shown on the graph.

Figure 5. A schematic diagram of the possible mechanisms involved in the peripheral blood changes and their relationship to oxidative stress.

V. Varney et al.

101

stress and the generation of oxygen free radicals is thought to play a role in the pathogenesis and progression of the disease. This is well described in bleomycin models where superoxide and hydroxyl radicals are generated [22] [23].

Our findings show a pattern of peripheral blood abnormalities in patients with lung fibrosis, irrespective of their subtype which is not seen in the age and sex matched controls. An association between IPF and EBV infection was one of the original hypotheses as a causal agent for IPF. EBV is a herpesvirus prevalent in man with a number of disease associations which include B cell and Burkitts lymphoma, Hodgkin’s disease and nasopharyngeal carcinoma [24]. Interestingly the observed blood changes in IPF are similar to those that may be seen in infec-tive mononucleosis (glandular fever). Furthermore, IPF is associated with enlargement of mediastinal lymph nodes, the cause of which is not been ex-plained. EBV has been shown to stimulate the fibrotic cytokine TGF-beta-1 and epidermal-mesenchymal transition which is associated with IPF [25]. From our selected fibrosis patients, all showed antibody evidence of past exposure to EBV but none were positive for active EBV copy numbers in their white blood cells to suggest current active viral replication. A previous study in IPF has shown rear-ranged EBV genomes (termed WZhet) in 61% lung tissue from IPF patients and 59% of IPF buffy coats while only 4% of healthy blood donors show such a re-arrangement, suggesting an association of WZhet with IPF patients [26].

The published literature on lung oxidative stress could also explain our find-ings in the absence of active EBV and link them to abnormalities in the glu-tathione pathways particularly affecting red cell size and GGT levels (Figure 5). Glutathione metabolism and its role in IPF have gained interest through the NAC studies. Glutathione catabolism is up-regulated in response to oxidants and cytokines [27] [28], and has an important role in protecting membranes from free radical damage [29]. If mitochondrial glutathione is depleted, perme-abilization and death can occur [30]. GGT is a membrane bound enzyme linked to the breakdown of glutathione. The prevention of bleomycin induced lung fi-brosis in GGT-/-deficient mice may be related to maintained glutathione levels which reduce apoptosis and fibrosis [17]. This has interesting links to our ob-servations where cotrimoxazole treatment and clinical improvement occurred with a fall in serum GGT. Since glutathione homeostasis is disturbed in IPF, it may indicate that GGT is pro-inflammatory in man also.

Raised serum GGT > 50 U/L is also reported in relapsing cryptogenic organ-ising pneumonias, without evidence of cholelithiasis or liver disease [31] [32], and here chronic lung injury and fibrosis can also occur.

The elevation of MCV may reflect membrane oxidative stress and lipid per-oxidation in a similar fashion to liver disease [33] and be connected with the raised Ethane levels described in IPF [34] [35]. Red cells are more vulnerable to free radicals due to their iron content compared to white blood cells [36]. Meas-urements of Ethane are not routinely available, but are elevated in IPF and are suggested to be valuable as markers of lipid membrane peroxidation [34].

Monocytes are intimately involved with wound repair and fibrosis. M2 ma-

V. Varney et al.

102

crophages, are the counterparts to TH2 cells and generally anti-inflammatory and produce growth factors including TGF beta-1 and platelet derived growth factors [37]. TGF beta-1 enhances fibroblast differentiation and the synthesis of collagens by myofibroblasts, with high levels reported in honeycomb lung [38] [39] [40]. Monocytes themselves can generate oxygen free radicals to regulate other inflammatory cells [39]. Oxidative stress disturbs normal cytokine regula-tion and promotes fibrogenesis [40] [41] [42]. In human and animal models of fibrotic organ injury, the site of tissue damage triggers the recruitment of mo-nocytes often via granulocyte monocyte colony stimulating factor [43]. Blocking of these monocytes in animal studies arrests fibrotic processes [44] [45].

The reduced peripheral monocyte count seen with cotrimoxazole treatment could be due to anti-inflammatory effects on the lung cytokine milieu. This could reduce monocytes recruitment and monocyte derived ROS [46] [47].

The linear relationship between the peripheral monocyte count and the serum TGF beta-1 levels in the pilot study are interesting due to the potential signifi-cance of this cytokine. After cotrimoxazole treatment, both values fell but the relationship remained the same with an increase of 600 pg/ml of TGF-beta-1 for every increase in monocyte count of 0.1 × 109. One possible explanation is that TGF beta-1 is derived from the blood monocytes. This is an important fibrotic cytokine, linked to the production of ROS, with resistance to apoptosis and epithelial-mesenchymal differentiation [48] [49] [50].

Cotrimoxazole in IPF has gained further attention recently following the pub-lication of the TIPAC study [51]. A double blind randomised placebo controlled study of cotrimoxazole or placebo added to standard care medication (predni-solone, azathioprine, NAC and mycophenolate mofetil); showed no significant improvement in forced vital capacity, transfer factors or 6minute walk test, but a 5-fold reduction in all cause mortality in those adherent to treatment. There were improved health related scores with reduced sudden exacerbations.

The study did not address the mechanism, but the possible antibacterial properties of cotrimoxazole were raised. Bacterial infection in newly presenting cases of IPF has been examined by bronchoscopy with quantitative culture to establish bacterial levels in the lower airways [52] [53]. Pathogens were isolated in 36% of IPF patients but not in healthy controls. The authors questioned whether unrecognised colonisation is a determinant in this disease [54].

Sulphonamides such as sulfamethoxazole and dapsone are structural ana-logues of para-aminobenzoic acid, and competitively inhibit the first step in the synthesis of folic acid (dihydro-folate) affecting growth and replication of micro- organisms. Trimethoprim blocks the next step in folic acid metabolism affecting nucleic acid and protein synthesis [55]. Cotrimoxazole has good penetration to the bronchial secretions. It is active against Pneumocystis jiroveci, streptococcus pneumoniae and a wide range of aerobic gram negative microorganisms [55]. It is used long term in HIV disease and organ transplantation with a good safety record and no significant problems of bacterial resistance/overgrowth.

Dapsone shares cotrimoxazole’s sulphonamide ring with similar actions on

V. Varney et al.

103

folic acid synthesis and bacteria. Detailed studies have shown Dapsone to also have extensive actions on the immune system and oxidative stress. It prevents the formation of oxygen free radicals at sites of infection by inhibiting myelop-eroxidase in mononuclear cells and neutrophils through direct effects on these cells [56]. Dapsone has been shown to block lipid peroxidation and maintain glutathione levels in animal models of neurotoxicity [57]. Detailed in-vitro stud-ies show substantial anti-oxidative properties attributed largely to inhibition of monocyte and neutrophil ROS production [58], but comparable studies on co-trimoxazole have not been conducted.

5. Conclusion

These findings suggest that the blood abnormalities noted in lung fibrosis pa-tients could fit with oxidative stress leading to monocyte recruitment, rather than active EBV disease. The effect of cotrimoxazole on mononuclear cells may be similar to that described for dapsone, with reductions in oxygen free radicle generation reducing lipid peroxidation and the fibrotic cytokine TGF beta-1. This reduction in oxidative stress may explain the reduced mortality from sud-den exacerbations of IPF seen in patient on regular cotrimoxazole. Further stu-dies of cotrimoxazole effects on mononuclear cells and oxidative stress along with exhaled ethane levels are indicated and may increase the understanding of IPF.

Acknowledgements

1) Peel Medical Research Trust, 40 Tower hill, London. EC3N 4DX, for sup-porting the pilot study medication costs, but had no involvement in the study design, data analysis or preparation of this manuscript.

2) Irina Chisster, Statistician at Kings College Hospital, London for help with data analysis.

3) Annette Davies, preoperative clinic sister at Epsom and St Helier NHS Trust.

References [1] Varney, V.A., Parnell, H.M., Salisbury, D.T., et al. (2008) A Double Blind Random-

ised Placebo Controlled Pilot Study of Oral Cotrimoxazole in Advanced Fibrotic Lung Disease. Pulmonary Pharmacology & Therapeutics, 21, 178-187. https://doi.org/10.1016/j.pupt.2007.02.001

[2] Kinnula, V.L. and Myllärniemi, M. (2008) Oxidant-Antioxidant Imbalance as a Po-tential Contributor to the Progression of Human Pulmonary Fibrosis. Antioxidants & Redox Signaling, 10, 727-738. https://doi.org/10.1089/ars.2007.1942

[3] Cantin, A.M., Hubbard, R.C. and Crystal, R.G. (1989) Glutathione Deficiency in the Epithelial Lining Fluid of the Lower Respiratory Tract in Idiopathic Pulmonary Fi-brosis. American Review of Respiratory Disease, 139, 370-372. https://doi.org/10.1164/ajrccm/139.2.370

[4] Thomas, J.P., Maiorino, M., Ursini, F. and Girotti, A.W. (1990) Protective Action of Phospholipid Hydroperoxide Gluthathione Peroxidase against Membrane-Damag- ing Lipid Peroxidation. The Journal of Biological Chemistry, 265, 454-461.

[5] Raes, M., Michiels, C. and Remacle, J. (1987) Comparative Study of the Enzymatic

V. Varney et al.

104

Defense Systems against Oxygen Derived Free Radicles: The Key Role of Gluta-thione Peroxidase. Free Radical Biology & Medicine, 3, 3-7.

[6] Flohe, L. (1988) Glutathione Peroxidase. In: Simic, M.G., Taylor, K.A., Ward, J.F. and von Sonntag, C., Eds., Oxygen Radicals in Biology and Medicine, Basic Life Sci- ences Vol. 49, Springer, USA, 663-668. https://doi.org/10.1007/978-1-4684-5568-7_104

[7] Demedts, M., Behr, J., Buhl, R., et al. (2005) High-Dose Acetylcysteine in Idiopathic Pulmonary Fibrosis. The New England Journal of Medicine, 353, 2229-2242. https://doi.org/10.1056/NEJMoa042976

[8] The Idiopathic Pulmonary Fibrosis Research Network (2014) Randomised Trial of Acetyl Cysteine in Idiopathic Pulmonary Fibrosis. The New England Journal of Me- dicine, 370, 2093-2101. https://doi.org/10.1056/NEJMoa1401739

[9] Memesanszky, E. and Lott, J.A. (1985) Gamma Glutamyltransferase and Its Isoen- zymes. Clinical Chemistry, 31, 797-803.

[10] Bellini, M., Tumino, E., Giordani, R., et al. (1997) Serum Gamma-Glutamyl Trans- petidase Isoforms in Alcoholic Liver Disease. Alcohol and Alcoholism, 32, 259-266. https://doi.org/10.1093/oxfordjournals.alcalc.a008265

[11] Moss, D.W., Echetebu, Z.O., Whitaker, O.J., et al. (1982) Multiple Forms of Gamma Glutamyl Transferase and Their Clinical Significance. Advance Biol Pharm, 3, 41- 45.

[12] Rogers, A.J., Brasch-Andersen, C. and Lonita-Laza, A. (2009) The Interaction of Glutathione S-Transferase M1-Null Variants with Tobacco Smoke Exposure and the Development of Childhood Asthma. Clinical & Experimental Allergy, 39, 1721- 1729. https://doi.org/10.1111/j.1365-2222.2009.03372.x

[13] Comporti, M. (1985) Biology of Disease: Lipid Peroxidation and Cellular Damage in Toxic Liver Injury. Laboratory Investigation, 53, 599-623.

[14] Akkus, I., Gultekin, F., Akoz, M., et al. (1997) Effect of Moderate Alcohol Intake on Lipid Peroxidation in Plasma, Erythrocytes and Leucocyte and on Some Antioxi-dant Enzymes. Clinica Chimica Acta, 266, 141-147. https://doi.org/10.1016/S0009-8981(97)00135-6

[15] Sacchetti, L., Giuseppe, C. and Salvatore, F. (1989) Electrophoretic Behaviour and Partial Characterization of Disease-Associated Serum Forms of Gamma Glutamyl-transferase. Electrophoresis, 10, 619-627. https://doi.org/10.1002/elps.1150100815

[16] Bently, A.R., Emrani, P. and Cassano, P.A. (2008) Genetic Variation and Gene Ex-pression in Antioxidant Related Enzymes and Risk of COPD: A Systematic Review. Thorax, 63, 956-961. https://doi.org/10.1136/thx.2007.086199

[17] Pardo, A., Ruiz, V., Arreola, J.L., et al. (2003) Bleomycin-Induced Pulmonary Fibro- sis Is Attenuated in Gamma-Glutamyl Transpeptidase-Deficient Mice. American Journal of Respiratory and Critical Care Medicine, 167, 925-932. https://doi.org/10.1164/rccm.200209-1007OC

[18] Shaw, L.M., Strømme, J.H., Loudon, J.L. and Theodosen, L. (1983) IFCC Methods for the Measurement of Catalytic Concentration of Enzymes. Part 4 IFCC Method for Gamma Glutamyltransferase. Journal of Clinical Chemistry & Clinical Bioche- mistry, 21, 633-646. www.atsjournals.org

[19] Bradley, B., et al. (2008) Interstitial Lung Disease Guideline: The British Thoracic Society in Collaboration with the Thoracic Society of Australia and New Zealand and the Irish Thoracic Society. Thorax, 63, v1-v58.

[20] Raghu, G., et al. (2011) An Official ATS/ERS/JRS/ALAT Statement: Idiopathic Pul- monary Fibrosis: Evidence-Based Guidelines for Diagnosis and Management. Ame-

V. Varney et al.

105

rican Journal of Respiratory and Critical Care Medicine, 183, 788-824. https://doi.org/10.1164/rccm.2009-040GL

[21] Hansell, D.M. and Wells, A.U. (1996) CT Evaluation of Fibrosing Alveolitis-Appli- cations and Insights. Journal of Thoracic Imaging, 11, 231-249. https://doi.org/10.1097/00005382-199623000-00001

[22] Gabrielli, A., Svegliati, S., Moroncini, G. and Amico, D. (2012) New Insights into the Role of Oxidative Stress in Scleroderma Fibrosis. The Open Rheumatology Jour- nal, 6, 87-95. https://doi.org/10.2174/1874312901206010087

[23] Yoshizaki, A., Yanaba, K., Ogawa, A., et al. (2011) The Specific Free Radical Scaven- ger Edaravone Suppresses Fibrosis in Tight-Skin and Bleomycin-Induced Mouse Model of Systemic Sclerosis. Arthritis & Rheumatology, 63, 3086-3097. https://doi.org/10.1002/art.30470

[24] Egan, J.J., Stewart, J.P., Hasleton, P.S., Arrand, J.R., Carroll, K.B. and Woodcock, A.A. (1995) Epstein-Barr Virus Replication within Pulmonary Epithelial Cells in Cryptogenic Fibrosing Alveolitis. Thorax, 50, 1234-1239. https://doi.org/10.1136/thx.50.12.1234

[25] Sides, M.D., Klingsberg, R.C., Shan, B., Gordon, K.A., Nguyen, H.T., Lin, Z., Taka-hashi, T., Flemington, E.K. and Lasky, J.A. (2011) The Epstein-Barr Virus Latent Membrane Protein-1 and Transforming Growth Factor—β1 Synergistically Induce Epithelial—Mesenchymal Transition in Lung Epithelial Cells. American Journal of Respiratory Cell and Molecular Biology, 44, 852-862.

[26] Kelly, B.G., Lok, S.S., Hasleton, P.S., Egan, J.J. and Stewart, J.P. (2002) A Rearranged Form of Epstein-Barr Virus DNA Is Associated with Idiopathic Pulmonary Fibrosis. American Journal of Respiratory Cell and Molecular Biology, 166, 510-513.

[27] Coker, R.K. and Laurent, G.J. (1998) Pulmonary Fibrosis: Cytokines in Balance. European Respiratory Journal, 11, 1218-1221. https://doi.org/10.1183/09031936.98.11061218

[28] Jaya, D.S., Augstine, J. and Menon, V.P. (1993) Role of Lipid Peroxidation, Gluta-thione and Antiperoxidative Enzymes in Alcohol and Drug Toxicity. Indian Journal of Experimental Biology, 31, 453-459.

[29] Valko, M., Leibfritz, D., Moncot, J., Cronin, M.T.D., Mazur, M. and Telser, J. (2007) Free Radicals and Antioxidants in Normal Physiological Functions and Human Di- sease. The International Journal of Biochemistry & Cell Biology, 39, 44-48.

[30] Mates, J.M., Sequra, J.A., Alonso, F.S. and Marquez, J. (2008) Intracellular Redox Status and Oxidative Stress: Implications for Cell Proliferation, Apoptosis Granulo-genesis. Archives of Toxicology, 82, 273-299. https://doi.org/10.1007/s00204-008-0304-z

[31] Lazor, R., Vandevenne, A., Pelletier, A., et al. (2000) Cryptogenic Organizing Pneu- monia. Characteristics of Relapses in a Series of 48 Patients. American Journal of Respiratory and Critical Care Medicine, 162, 571-577. https://doi.org/10.1164/ajrccm.162.2.9909015

[32] Schlesinger, C. and Koss, M.N. (2005) The Organising Pneumonias: An Update and Review. Current Opinion in Pulmonary Medicine, 11, 422-430. https://doi.org/10.1097/01.mcp.0000175521.41729.07

[33] Lindi, C., Montorfano, G. and Marciani, P. (1998) Rat Erythrocyte Susceptibility to Lipid Peroxidation after Chronic Ethanol Intake. Alcohol, 16, 311-316. https://doi.org/10.1016/S0741-8329(98)00020-2

[34] Paredi, P., Kharitonov, S.A. and Leak, D. (2000) Exhaled Ethane, a Marker of Lipid Peroxidation. American Journal of Respiratory and Critical Care Medicine, 162, 369-373. https://doi.org/10.1164/ajrccm.162.2.9909025

V. Varney et al.

106

[35] Kanoh, S., Kobayashi, H. and Motoyoshi, K. (2005) Exhaled Ethane: An in Vivo Biomarker of Lipid Peroxidation in Interstitial Lung Diseases. Chest, 128, 2387- 2392.

[36] Gatti, P., Viani, P., Cervato, G., et al. (1993) Effects of Alcohol Abuse: Studies on Human Erythrocyte Susceptibility to Lipid Peroxidation. Biochemistry and Mole-cular Biology International, 30, 807-917.

[37] Koli, K., Myllarniemi, M., Keski-Oja, J. and Kinnula, V.L. (2008) Transforming Growth Factor Beta Activation in the Lung: Focus on Fibrosis and Reactive Oxygen Species. Antioxidants & Redox Signaling, 10, 333-342.

[38] Knobloch, J., Peters, H., Jungck, D., et al. (2009) TNFα-Induced GM-CSF Release from Human Airway Smooth Muscle Cells Depends on Activation of an ET-1 Auto- regulatory Positive Feedback Mechanism. Thorax, 64, 1044-1052. https://doi.org/10.1136/thx.2008.111047

[39] Mosser, D.M. (2003) The Many Faces of Macrophage Activation. Journal of Leuko-cyte Biology, 73, 209-212. https://doi.org/10.1189/jlb.0602325

[40] Gratchev, A., Guillot, P., Hakiy, N., et al. (2001) Alternately Activated Macrophages Differentially Express Fibronectin and Its Splice Variants and the Extracellular Ma-trix Protein βIG-H3. Scandinavian Journal of Immunology, 53, 386-392. https://doi.org/10.1046/j.1365-3083.2001.00885.x

[41] Murray, P. and Wynn, T. (2011) Protective and Pathogenic Functions of Macro- phages Subsets. Nature Reviews Immunology, 11, 723-737. https://doi.org/10.1038/nri3073

[42] Keerthisingam, C.B., Jenkins, G., Harrison, N.K., et al. (2001) Cyclooxygenase-2 De- ficiency Results in a Loss of the Anti-Proliferative Response to Transforming Growth Factor-β in Human Fibrotic Lung Fibroblasts and Promotes Bleomycin- Induced Pulmonary Fibrosis in Mice. The American Journal of Pathology, 158, 1411-1422. https://doi.org/10.1016/S0002-9440(10)64092-8

[43] Nathan, C. and Ding, A. (2010) Nonresolving Inflammation. Cell, 140, 871-882. https://doi.org/10.1016/j.cell.2010.02.029

[44] Lin, S.H., Castano, A.P., Nowlin, B.T., Lupher, M.L. and Duffield, J.S. (2009) Bone Marrow LY6Chigh Monocytes Are Selectively Recruited to Injured Kidney and Di- fferentiate into Functionally Distinct Populations. The Journal of Immunology, 183, 6733-6743.

[45] Duffield, J.S., Forbes, S.J., Constandinou, M., et al. (2005) Selective Depletion of Macrophages Reveals Distinct, Opposing Roles during Liver Injury and Repair. Journal of Clinical Investigation, 115, 56-56. https://doi.org/10.1172/JCI200522675

[46] Rahman, I. and Macnee, W. (2000) Oxidative Stress and Regulation of Glutathione in Lung Inflammation. European Respiratory Journal, 16, 534-554. https://doi.org/10.1034/j.1399-3003.2000.016003534.x

[47] Kinnula, V.L. and Crapo, J.D. (2009) Superoxide Dismutases in the Lung and Hu-man Lung Disease. American Journal of Respiratory and Critical Care Medicine, 179, 542-548.

[48] Willis, B.C., Liebler, J.M., Luby-Phelps, K., Nicholson, A.G., Crandall, E.D., du Bois, R.M. and Borok, Z. (2005) Induction of Epithelial-Mesenchymal Transition in Al-veolar Epithelial Cells by Transforming Growth Factor β1: Potential Role in Idi-opathic Pulmonary Fibrosis. The American Journal of Pathology, 166, 1321-1332. https://doi.org/10.1016/S0002-9440(10)62351-6

[49] Sheppard, D. (2006) Transforming Growth Factor β1: A Central Modulator of Pul-monary and Airway Inflammation and Fibrosis. Proceedings of the American Tho-

V. Varney et al.

107

racic Society, 3, 413-417. https://doi.org/10.1513/pats.200601-008AW

[50] Biemacka, A., Dobaczewski, M. and Frangogiannis, N.G. (2011) TGF-β Signaling in Fibrosis. Growth Factors, 29, 196-202. https://doi.org/10.3109/08977194.2011.595714

[51] Shulgina, L., Cahn, A.P., Chilvers, E.R., Parfrey, H., Clark, J.J., Wilson, E.C.F., Twentyman, O.P., Davison, A.G., Curtin, J.J., Crawford, M.B. and Wilson, A.M. (2013) Treating Idiopathic Pulmonary Fibrosis with the Addition of Co-Trimoxa- zole: A Randomised Controlled Study. Thorax, 68, 155-162. https://doi.org/10.1136/thoraxjnl-2012-202403

[52] Molyneaux, P.L. and Maher, T.M. (2013) The Role of Infection in the Pathogenesis of Idiopathic Pulmonary Fibrosis. European Respiratory Review, 22, 376-381. https://doi.org/10.1183/09059180.00000713

[53] Richter, A.G., Stockley, R.A., Harper, L. and Thickett, D.R. (2009) Pulmonary Infec-tion in Wegener Granulomatosis and Idiopathic Pulmonary Fibrosis. Thorax, 64, 692-697. https://doi.org/10.1136/thx.2008.110445

[54] Harrison, N.K. (2009) Pulmonary Infection in Wegener’s Granulomatosis and Idi-opathic Pulmonary Fibrosis. Thorax, 64, 647-649. https://doi.org/10.1136/thx.2009.115089

[55] Kaplan, S.A. (1973) Pharmacokinetic Profile of Trimethoprim-Sulfamethoxazole in Man. The Journal of Infectious Diseases, 218, S547-S555. https://doi.org/10.1093/infdis/128.Supplement_3.S547

[56] Bozeman, P.M., Learn, D.B. and Thomas, E.L. (1992) Inhibition of the Human Leu- kocyte Enzymes Myeloperioxidase and Eosinophils Peroxidase by Dapsone. Bioche- mical Pharmacology, 44, 553-563.

[57] Akagracia, M., Monroy-Noyola, A., Osorio-Rico, L., Kravzov, J. and Rios, C. (1994) Dapsone Attenuates Kainic Acid-Induced Seizures in Rats. Neuroscience Letters, 176, 52-54.

[58] Worsel, G. and Blasum, C. (2014) Dapsone in Dermatology and beyond. Archives of Dermatological Research, 306, 103-124. https://doi.org/10.1007/s00403-013-1409-7

V. Varney et al.

108

Abbreviations

GSH Glutathione GST Glutathione-S-transferase MCV Mean cell volume GGT Gamma glutamyl transferase Mo Monocytes in peripheral blood HRCT High resolution CT scan UIP Usual interstitial pneumonitis NSIP Non specific interstitial pneumonitis TGF beta-1 Transforming growth factor beta-1 ROS Reactive oxygen species NAC N acetyl cysteine GMCSF Granulocyte monocyte colony stimulating factor EBV Epstein barr virus IPF Idiopathic pulmonary fibrosis

Submit or recommend next manuscript to SCIRP and we will provide best service for you:

Accepting pre-submission inquiries through Email, Facebook, LinkedIn, Twitter, etc. A wide selection of journals (inclusive of 9 subjects, more than 200 journals) Providing 24-hour high-quality service User-friendly online submission system Fair and swift peer-review system Efficient typesetting and proofreading procedure Display of the result of downloads and visits, as well as the number of cited articles Maximum dissemination of your research work

Submit your manuscript at: http://papersubmission.scirp.org/ Or contact pp@scirp.org